Biomedical subjects
Publications and source records attributed to S Hyman.
Although rodents are nocturnal, their behavior is usually tested during the day. The authors present the results of a preliminary study, which suggest that altering the animals' day:night cycle might be the key to eliminating the need for food or water deprivation prior to testing.
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Iodide uptake by the sodium/iodide symporter (NIS) in thyrocytes is essential for thyroid hormone production. Reduced NIS activity has been reported in thyroid diseases, including thyroid cancer and congenital hypothyroidism. The study of iodide uptake in thyrocytes has been limited by the availability of appropriate in vitro models. A new culture technique was recently developed that allows normal human thyroid primary culture cells to grow as monolayer cells and express differentiated functions for more than 3 months. We used this technique to study the effect of follicle formation and TSH on iodide uptake in these cells. Iodide uptake by the cells grown in monolayer was very low. Follicle formation was induced from monolayer cells, and electron micrographs demonstrated cell polarity in the follicles. No significant increase in iodide uptake was observed after TSH treatment of cells in monolayer or when follicle formation was induced without TSH. TSH stimulation of follicles, however, significantly increased iodide uptake ( approximately 4. 4-fold; P<0.001). Compared with iodide uptake in monolayers, the combination of follicle formation and TSH treatment stimulated iodide uptake synergistically to 12.0-fold (P<0.001). NIS messenger RNA (mRNA) and protein levels were almost the same in both monolayer cells and follicles. TSH treatment of monolayers and follicles produced significant (P<0.05) stimulation of mRNA ( approximately 4. 8- and approximately 4.3-fold respectively) and protein ( approximately 6.8- and 4.9-fold respectively). TSH stimulated NIS protein levels in both monolayer and follicles, however, stimulation of functional iodide uptake was only seen with TSH stimulation of follicles. The function of NIS may involve post-transcriptional events, such as intracellular sorting, membrane localization of NIS or another NIS regulatory factor. Polarized functions, such as iodide efflux into follicular lumina, may also contribute to the increased iodide concentration after follicle formation.
The objective of this article is to provide the reader with an update of some of the BBB research highlights which have occurred in recent times, and to review the impact and contributions of immunogold electron microscopic studies on our understanding of the brain capillary endothelium. Glucose and monocarboxylic acids are two small molecules which this review will focus upon; and advances in immunogold characterization of the GLUT1 glucose transporter and the MCT1 and MCT2 monocarboxylic acid nutrient transporters will be discussed. Human serum albumin is chosen as a representative large molecule, and it has recently been shown that immunogold identification of this protein can serve as an indicator of compromised BBB function in a variety of pathophysiological conditions.
Hydrocephalus is a common and potentially lethal condition in children that results from an imbalance between absorption and production of cerebral spinal fluid (CSF). Silastic shunts are inserted to drain excess CSF, but they are prone to a number of problems, and at times may be unreliable and ineffective. This study examines the physiological basis of a pedicled omental transfer to the brain as a functional conduit for CSF in an experimentally induced hydrocephalic rabbit model. The ability of the omentum to transport CSF from the subarachnoid space was tested using radioactive tracer substances: radio-iodinated serum albumin (125I-RISA), chromium 51-ethylenediaminetetraacetate (51Cr-EDTA), and technetium 99m (99mTc) glucoheptonate. Immediate ability of exteriorized omentum to transport artificial CSF, as well as transposed omental transport of subarachnoid CSF at 1 month, were examined. Nuclear scan measurements were correlated with clinical observation and a double-blind histological analysis with trichrome and hematoxylin-eosin stain. Exteriorized omentum rapidly absorbed 99mTc glucoheptonate-labeled artificial CSF, with a rapid appearance in the systemic circulatory and urinary systems. Transposed omentum to the brain in animals with artificially created hydrocephalus showed evidence of CSF-labeled 125I-RISA and 51Cr-EDTA absorption in those animals demonstrating histologically viable omentum.
Immunogold electron microscopy was used to analyze and quantify the Glut1 glucose transporter in brain tissue from five patients undergoing surgery for treatment of seizures. Samples were prepared from two different regions of each resection: (1) the most actively spiking epileptogenic site, and (2) the least actively spiking region, as indicated by intraoperative EEG monitoring. Two configurations of endothelial cell Glut1 were observed. About one half of the capillary profiles examined displayed abundant Glut1 immunoreactivity on both luminal and abluminal endothelial membranes. In the remainder of the profiles, reduced Glut1 labeling was seen, but adjacent erythrocyte membranes remained highly Glut1 immunoreactive, suggesting that reduced endothelial Glut1 reactivity was not attributable to method artifacts. Immunogold studies using antisera to human glial fibrillary acidic protein and human serum albumin demonstrated increased quantities of these two epitopes in the extravascular regions in which more EEG spiking activity had been demonstrated. These observations were consistent with the hypotheses that capillary integrity was more compromised, and gliosis was quantitatively increased, in the more actively spiking region of the resection. Altered glucose transporter activity in the blood-brain barrier was characterized by a bimodal Glut1 distribution in which the smaller (type B) endothelial cells displayed low Glut1 immunoreactivity, whereas adjacent (and even contiguous) larger (type A) endothelial cells showed 5- to 10-fold greater expression of membrane Glut1 transporter protein. Because this transporter facilitates glucose entry to the brain, small pericapillary volumes of brain tissue may have quite different concentrations of glucose. We hypothesize that in complex partial seizures and other forms of brain insult, an alteration of blood-brain barrier Glut1 glucose transporter activity is indicated by the appearance of these two subpopulations of endothelial cells. In comparison with previous studies of human brain capillaries in hemangioblastoma and brain injury, endothelial Glut1 density was apparently reduced (interictally) in affected temporal lobes of patients with complex partial seizures.
The objective of the present study was to define the cellular location of the Glut1 glucose transporter in the primate choroid plexus. Immunogold electron microscopy indicated that Glut1 epitopes were associated primarily with choroid plexus endothelial cells. Digitized analyses of electron microscopic images provided quantitative estimates of the relative number of Glut1 glucose transporter epitopes on luminal and abluminal endothelial cell membranes within the choroid plexuses. We recorded a high density of Glut1 in the microvascular endothelium of primate choroid plexus, which was consistent in vervet monkeys (5-10 Glut1 gold particles per micrometer of endothelial cell plasma membrane), as well as in baboons (5-20 Glut1 gold particles per micrometer of capillary plasma membrane). In the baboon choroid plexus, we observed that perivascular cells (presumed to be pericytes) were also Glut1-positive, but with substantially reduced activity compared with endothelial cells. Occasional Glut1-immunogold particles were also seen in the basolateral membranes of the choroid plexus cuboidal cells. Light microscopic immunocytochemistry confirmed the abundance of Glut1 immunoreactivity in choroid plexus endothelial cells of vervet monkeys and baboons. A similar pattern was observed in surgically resected human choroid plexus, suggesting differences between primates, including humans and laboratory animals. The only difference was that erythrocytes within the human choroid plexus exhibited a florid Glut1-positive response, but were weakly immunoreactive in nonhuman primates. The observation of high glucose transporter densities in choroid plexus endothelial cells is consistent with the suggestion that choroidal epithelia and capillaries provide a metabolic work capability for maintaining ionic gradients and secretory functions across the blood-CSF barriers.
Cellular distribution of the Glut1 glucose transporter in normal primate brains was analyzed by immunogold electron microscopy. Two configurations of endothelial Glut1 glucose transporter (high and low density capillaries) have been found in resections of traumatically injured and epileptogenic human brain; the objective of the present study was to ascertain whether these same 2 capillary populations, expressing high and low glucose transporter densities, were the common configuration in normal brain. The relative numbers of Glut1 glucose transporter-associated gold particles on luminal and abluminal endothelial cell membranes were determined within the cerebral cortex of several normal, nonhuman primates. Low Glut1 densities were seen in brain endothelia of both the rhesus and squirrel monkey cortex, with slightly greater quantities of Glut1 in vervet monkey cortices. The Glut1 transporter was most highly expressed in the baboon cortex, approaching the concentrations seen in human brains. In the rhesus, squirrel, and vervet monkeys, Glut1 concentrations were greater on the abluminal than luminal capillary membranes. In contrast, mean luminal membrane Glut1 concentrations were greater in baboons, resembling the distribution seen in the human brain. Brain regional differences in transporter concentration were seen in comparing membrane densities in the baboon cortex (approximately 15 Glut1-gold particles per micrometer), hippocampus (approximately 12 Glut1 gold particles per micrometer), cerebellum (approximately 6 Glut1-gold particles per micrometer), and retinal microvasculature (approximately 20 Glut1-gold particles per micrometer). We conclude that a single, uniform Glut1 distribution characterizes brain capillaries of normal nonhuman primates, and hypothesize that the presence of high and low density glucose transporter endothelial cells (seen in human traumatic injury and seizure resections) represents a pathologic response to brain insult.
The objective of the present study was to examine the apparent work capacity of one of the two separate membrane systems (the blood-cerebrospinal fluid barrier) that isolate the mammalian brain extracellular fluid (and cerebrospinal fluid, CSF) from plasma. Digitized analyses of electron-microscopic images provided estimates of mitochondrial volumes, which were expressed as a percentage of the cell cytoplasm. We recorded a high mitochondrial content of 12-15% in the cuboidal epithelium of primate choroid plexus, which was consistent in vervet, rhesus, and squirrel monkeys, as well as in baboons. Similarly high mitochondrial contents were observed in the rabbit, rat, and mouse choroid plexus. It has been postulated that the high mitochondrial content of brain endothelium is associated with maintaining the ionic gradients within the central nervous system. We observed that the mitochondrial content of the choroid plexus (where CSF is produced) was slightly higher than in (prior measurements of) the blood-brain barrier (BBB). In addition, surface areas at the apical borders of the choroid plexus epithelia (where the Na+K+ATPase activity has been localized) were increased 7- to 13-fold over the basal borders, in the primate species examined. The observation of high mitochondrial volumes in choroid plexus cells is consistent with the suggestion that increased mitochondrial densities seen in choroidal epithelia and BBB capillaries provide a metabolic work capability for both secretory activities and maintaining ionic gradients across blood-CSF barriers.
The principal glucose transporter at the blood-brain barrier (BBB) is the Glut1 isoform, and transporter density is believed to be an index of cerebral metabolic rate. In the present study, glucose transporter expression was studied in tissue resected 7-8 h after acute traumatic brain injuries in 2 patients. Light microscopic immunochemistry indicated a zone of complete loss of the Glut1 glucose transporter isoform in microvessel endothelial cells adjacent to sites of small vessel injury, concentrically surrounded by a narrow zone of variable Glut1, and distally surrounded by capillaries with typically immunoreactive endothelia in nondisrupted parenchyma. Variably reactive capillaries displayed alternating sectors of greatly reduced and highly reactive Glut1 density, suggesting a high density and low density of transporter activity in contiguous endothelial cells. Quantitative electron microscopic immunogold analyses demonstrated that the transporter was predominantly localized to the luminal and abluminal endothelial membranes, with lesser reactivity in cytoplasm; pericyte Glut1 was minimally above background levels. In endothelial sectors with reduced Glut1 transporter immunoreactivity, the luminal:abluminal ratio of Glut1 epitòpes was less than unity; while it is greater than unity in highly reactive endothelial cells. The number of Glut1-immunoreactive sites per micrometer of capillary membrane was not significantly different from previous reported Glut1 density in seizure resections, and about 2- to 3-fold higher than in human red cells. In the same tissue samples, qualitative immunogold electron microscopy of human serum albumin indicated leakage of this protein (MW 65,000) from the vascular space into pericapillary regions. Thus the high Glut1 density observed in capillaries from acutely injured brain occurs concomitantly with compromised barrier function.
The intracarotid injection method has been utilized to examine blood-brain barrier (BBB) glucose transport in hyperglycemic (4-6 days) mice. In anesthetized mice, Brain Uptake Indices were measured over a range of glucose concentrations from 0.010-50 mmol/l; glucose uptake was found to be saturable and kinetically characterized. The maximal velocity (Vmax) for glucose transport was 989 +/- 214 nmol.min-1.g-1. and the half-saturation constant estimated to be 5.80 +/- 1.38 mmol/l. The unsaturated Permeability Surface area product (PS) is = 171 + 8 microliters.min.-1.g-1. A rabbit polyclonal antiserum to a synthetic peptide encoding the 13 C-terminal amino acids of the human erythrocyte glucose transporter immunocytochemically confirmed the presence of the GLUT1 isoform in non-obese diabetic (NOD) mouse brain capillary endothelia. These studies indicate that a down-regulation of BBB glucose transport occurs in these spontaneously hyperglycemic mice; both BBB glucose permeability (as indicated by PS product) and transporter maximal velocity are reduced (in comparison to normoglycemic CD-1 mice), but the half-saturation constant remains unchanged.
The commonly held notion that Rett syndrome (RS) is a neurodegenerative disorder with normal early development was examined by an epidemiological survey and review of medical records and serial neurological and development evaluations. In some subjects, deviance from normal development was evident from the perinatal period, and gradually became more prominent with age. These findings are convincing when seen in conjunction with a reduction in velocity of brain growth, as early as 2-4 months of life, well before the recognition of gross neurological deficits. Neurodevelopmental evaluations provide no indication that there is progressive loss of adaptive behaviors, or communication skills to indicate a neurodegenerative process. Taken together with the known neuropathological and neurochemical changes in RS brain we hypothesize that RS is a neurodevelopmental disorder, which has a genetic basis, and affects subsets of neurons and their connections during a period of vigorous brain growth, when synapse formation and pruning are at a peak. Studies of mitochondrial (mt) DNA in brain to understand the genetic mechanisms underlying matrilineal inheritance in the few familial cases, and mt structural and enzyme deficiencies have been unrevealing to date.
The principal glucose transporter at the blood-brain barrier is Glut1, and GLUT1 expression is downregulated in high grade gliomas. In the present study, glucose transporter expression was studied in surgically resected hemangioblastoma tissue. Light microscopic immunochemistry indicated the high expression of the Glut1 glucose transporter isoform throughout the central vascular endothelium of this tissue. Glial fibrillary acidic protein (GFAP) was observed only at the tumor border, with no GFAP immunoreactivity in stromal cells, pericytes or endothelia in the central tumor regions. It is generally believed that more Glut1 is found in erythrocytes than any other cell, but quantitative electron microscopic immunogold analyses of Glut1-immunoreactive sites per micron of capillary membrane showed the Glut1 density in tumor endothelial membranes glucose transporter was 2-3-fold higher than in human red cells. In the same tissue samples, qualitative immunogold electron microscopy of human serum albumin indicated that this protein (MW 65,000) moved freely from the vascular space into pericapillary regions, confirming the leaky barrier characteristics of the hemangioblastoma. These studies show that Glut1 expression may be high in endothelia that are highly permeable and devoid of astroglial contacts. Thus, human cerebral hemangioblastomas may provide a novel system for studying the induction of Glut1 in the blood-brain barrier.
Blood-brain barrier (BBB) glucose transport rates were measured using the intracarotid injection method in newborn, 14-day-old suckling, 28-day-old weanling and adult rabbits, and compared with membrane transporter density. Light microscope immunochemistry confirmed the presence of the GLUT1 glucose transporter isoform in these rabbits. Quantitative electron microscopic immunogold analyses of GLUT1-immunoreactive sites per micrometer of capillary membrane indicated GLUT1 density increased with age, and correlated with in vivo measurements of Vmax. Maximal transport velocities (Vmax) of glucose transfer (an indicator of the activity and relative number of transporter proteins) increased significantly (P = 0.05) with age: in neonates Vmax = 0.61 mumol.min-1.g-1, in sucklings Vmax = 0.68 mumol.min-1.g-1, in weanlings Vmax = 0.88 mumol.min-1.g-1, and in adults Vmax = 1.01 mumol.min-1 g-1. Cerebral blood flow (CBF) rates, increased with age from 0.19 and 0.26 ml.min-1.g-1 in neonates and sucklings to 0.51 (weanlings) and 0.70 (adults) ml.min-1.g-1. Non-linear regression analyses indicated the half-saturation constant (Km) for glucose transport ranged from 13 mM in adult rabbits to 19 mM in 14-day-old sucklings: differences in Km were not significant. Age-related changes in the Permeability-Surface Area product (PS +/- S.E.) of both water and glucose were also seen. At all ages studied, the diffusion component (Kd) of glucose uptake was not distinguishable from zero. We conclude developmental up-regulation of the rabbit BBB glucose transporter is characterized by no changes in transporter affinity, and provide the first demonstration of increased membrane transporter proteins correlating with an age-related increase (65%) in glucose transporter maximal velocity.
Immunogold electron microscopy was used to examine human brain resections to localize the GLUT1 glucose transporter. The tissue examined was obtained from a patient undergoing surgery for treatment of seizures, and the capillary profiles examined had characteristics identical to those described previously for active, epileptogenic sites (confirmed by EEG analyses). A rabbit polyclonal antiserum to the full-length human erythrocyte glucose transporter (GLUT1) was labeled with 10-nm gold particle-secondary antibody conjugates and localized immunoreactive GLUT1 molecules in human brain capillary endothelia, with < 0.25% of the particles beyond the capillary profile. Erythrocyte membranes were also highly immunoreactive, whereas macrophage membranes were GLUT1-negative. The number of immunoreactive sites per capillary profile was observed to be 10-fold greater in humans than in previous studies of rat and rabbit brain capillaries. In addition, half of the total number of immunoreactive gold particles were localized to the luminal capillary membrane. We suggest that the blood-brain barrier GLUT1 glucose transporter is up-regulated in seizures, and this elevated transporter activity is characterized by increased GLUT1 transporters, particularly on the luminal capillary membranes. In addition, acute modulation of glucose transporter activity is presumed to involve translocation of GLUT1 from cytoplasmic to luminal membrane sites, demonstrable with quantitative immunogold electron microscopy.
The intracarotid injection method has been utilized to examine blood-brain barrier (BBB) glucose transport in normal mice, and after a 2-day fast. In anesthetized mice, cerebral blood flow (CBF) rates were reduced from 0.86 ml.min-1 x gm-1 in control to 0.80 ml.min-1 x gm-1 in fasted animals (p > 0.05). Brain Uptake Indices were significantly (p < 0.05) higher in fasted (plasma glucose = 4.7 mM) than control (plasma glucose = 6.5 mM) mice, while plasma glucose was significantly lower. The maximal velocity (Vmax) for glucose transport was 1562 +/- 303 nmoles.min-1 x g-1, and the half-saturation constant (Km =) 6.67 +/- 1.46 mM in normally fed mice. In fasted mice the Vmax was 2053 +/- 393 nmoles.min-1 x g-1 (p > 0.05), and the half-saturation constant (Km =) 7.40 +/- 1.60 mM (not significant, P > 0.05). A rabbit polyclonal antiserum to a synthetic peptide encoding the 13 C-terminal amino acids of the human erythrocyte glucose transporter (GLUT-1) immunocytochemically confirmed that the mouse brain capillary endothelial glucose transporter is a GLUT-1 transporter, and immunoreactivity was similar in brain endothelia from fed and fasted animals. In conclusion, after a 2-day fast in the mouse, we saw significant reductions in forebrain weight (7%), and plasma glucose levels (27%). Increased brain glucose extraction (25%, p < 0.05), and a 22% increase in the unsaturated permeability-surface area product (p < 0.05) was also observed.
Electron microscopy was used to quantitate blood-brain barrier (BBB) glucose transporters in newborn, 14-day-old suckling, 28-day-old weanling, and adult rabbits. A rabbit polyclonal antiserum to a synthetic peptide encoding the 13 C-terminal amino acids of the human erythrocyte glucose transporter (GLUT1) was labeled with 10-nm gold particle-secondary antibody conjugates and localized immunoreactive GLUT1 molecules in rabbit brain capillary endothelia. Three distinct populations of brain capillary profiles were identified in newborn rabbits: prepatent capillary buds, partially patent capillaries with highly amplified luminal membranes, and patent capillaries. Immunogold analyses indicated that the GLUT1 transporter abundance positively correlated with capillary developmental status. The mean number of gold particles per capillary profile increased at each developmental age examined, suggesting that developmental up-regulation of the BBB glucose transporter occurred in rabbits. GLUT1 immunoreactivity was three- to fourfold greater on the abluminal than luminal capillary membranes among all ages examined. Changes in the proportions of GLUT1 transporter were also seen, and possible reasons for the postnatal decrease in the percentage of cytoplasmic GLUT1 transporter are discussed. The numbers of cytoplasmic and membrane-associated immunogold particles increased with age. We conclude that regulatory modulations of BB glucose transport may be characterized by increases in BBB glucose transporter density with age and state of development. In addition, modulation of glucose transporter activity may be reflected by minor postnatal shifts of GLUT1 from cytoplasmic to membrane compartments, which can be demonstrated with quantitative immunogold electron microscopy.
Photodynamic therapy is a therapeutic modality long studies for its application to the treatment of malignant neoplasms. Recently, studies have suggested its potential use in the treatment of atherosclerosis. In this study, two atherosclerotic plaques were induced in the abdominal aortas of 35 rabbits. The animals then received Photofrin II (Quadralogic Technologies Inc., Pearl River, NY), a photosensitizer, at doses of 5 mg/kg and 2.5 mg/kg. After 48 hours, the plaques were irradiated by a fiberoptic connected to an argon ion laser. Fluency rates from 32 mW to 256 mW and energy doses from 1.6 to 60 joules were applied. Only one of the paired plaques was irradiated, the other remaining as a control. Four weeks after treatment, the vessels were assessed. Of 26 plaques treated with photodynamic therapy, 22 were no longer grossly visible, while the nine animals that received light irradiation but no Photofrin II all had visible plaque (P < 0.001). Studies of the vessel sections confirmed a reduction in intimal thickness from 0.74 +/- 0.15 mm in matched controls as compared with 0.51 +/- 0.13 mm in animals with treated plaques. There was a concomitant enlargement of the luminal diameter from 1.13 +/- 0.51 to 1.41 +/- 0.72. On the microscopic level, plaque reduction was most complete in the groups treated with 40 and 60 joules. Different fluency rates and drug dosages did not lead to differing outcomes. Our findings indicate that photodynamic therapy with dihematoporphyrin ether met our goal of reducing plaque size and may represent a means of treating atherosclerotic plaques.